Solution for conservation of living organs

ABSTRACT

The invention relates to medicine. The disclosed solution contains components in the following ratio, mM/l: 
     
         ______________________________________                                    
 
    
     Sodium chloride      100.0-117.0                                          
Potassium gluconate  12.0-17.0                                            
Calcium gluconate    1.2-2.0                                              
Magnesium sulphate    8.0-32.0                                            
Trihydroxymethylaminomethane                                              
                     2.0-3.0                                              
Local anesthetic     0.33-2.66                                            
Phenothiazine derivative                                                  
                     0.01-0.04                                            
Purine derivative    1.0-2.0                                              
Polyglucine          the balance                                          
______________________________________                                    
 
     The solution ensures successful conservation of living organs within up to 48 h.

FIELD OF ART

The present invention relates to medicine and, more specifically, to asolution for conservation of living organs.

BACKGROUND OF THE INVENTION

Known in the prior art are the methods for conservation of living organsand tissues, used in medicine and involving placing said organs andtissues into special conserving solutions followed by storing them underhypothermic conditions International Meeting on Phosphocreatine inCardiology and Cardiac Surgery, 1989, 14-15 Apr., Schiapparelli Searle(Pavia) P. Mastroberto, "Analisi degli effetti della creatina-fosfato inaggiunta a soluzione cardioplegica", p. 335-342.

Particular difficulties are encountered in conservation during surgicaloperations and transplantation of such an intensively functioning organas the heart which is characterized by intensive metabolic processes andenergy consumption during its contractions. If only a few years ago thesurgeons were capable of protecting the heart against total ischemia bymeans of cardioplegia in the course of the ist hour, at present thecooling and chemical cardioplegia suggested by the Royal St. ThomasInstitute and Hospital (Great Britain) ensures successful conservationof the heart for as long as 4 h. Plegisol (a commercial preparation)based on the cardioplegic solution worked out by the St. Thomas Hospitaland manufactured by the Abbott Laboratories, Chicago, U.S.A., is widelyused in clinical practice. Introduction of this preparation has improvedcellular protection during lengthy open-heart operations, contributed tofurther improvement of surgical techniques and extended the period ofconservation which is indispensable for heart transplantation(International Meeting on Phosphocreatine in Cardiology and CardiacSurgery, 1989, 14-15 April. Schiapparelli Searle (Pavial) D. J. Hearse"Protection of the Ischemic Myocardium: Cardioplegia", p. 173-183.

However, the conventionally recognized time of heart conservation (4 h)is insufficient for carrying it over large distances, to any point ofthe globe, where it has to be transplanted to a recipient. The increasedduration of conservation must be combined with improved quality andreliability of protecting the cells against ischemic and reperfusiondamage.

DISCLOSURE OF THE INVENTION

The main object of the invention lies in changing the qualitative andquantitative composition of the compound in order to evolve a solutionfor conservation of living organs featuring a longer period ofconservation and survival of cells thus ruling out their ischemic andreperfusion damage.

The object is attained by providing a solution for conservation oforgans containing sodium chloride, potassium salt, magnesium salt,calcium salt, pH stabilizer and solvent which contains additionally,according to the invention, a local anesthetic, phenothiazine and purinederivatives, potassium and calcium gluconates as substitutes for calciumsalts, magnesium sulphate as a substitute for magnesium salt,trihydroxymethylaminomethane as a pH stabilizer, and polyglucin as asolvent, in the following ratios of components, mM/l:

    ______________________________________                                        Sodium chloride      100.0-119.0                                              Potassium gluconate  12.0-17.0                                                Calcium gluconate    1.0-2.0                                                  Magnesium sulphate    8.0-32.0                                                Trihydroxymethylaminomethane                                                                       2.0-3.0                                                  Local anesthetic     0.33-1.66                                                Phenothiazine derivative                                                                           0.01-0.04                                                Purine derivative    1.0-2.0                                                  Polyglucine          the balance                                              ______________________________________                                    

The claimed solution extends considerably the conservation time ofliving organs up to 48 h.

BEST MODE OF CARRYING OUT THE INVENTION

The use of polyglucine for solvent increases radically the osmolarity ofthe solution (from 320 mOSm/l to 595 mOSm/l up) and prevents violentswelling of mitochondria and intracellular edema. The use of potassiumand calcium salts in the form of gluconates and magnesium salt in theform of magnesium sulphate instead of chlorides permits reducing thecontent of chlorine ions in the solution since the toxic effect of thelarge number of these ions on the membranes of the excitable cells, e.g.myocardine ones, is well known. The local anesthetic, for examplelidocaine, procaine, etc. is added into the solution for stabilizationof membranes. The phenothiazine derivative, e.g. ethacysine, is used forfixing the intracellular proteins of the calmoduline type and possessesa clearly pronounced antiarrhythmic and antiischemic effects. The purinederivative, for example inosine, is used to make up for the loss ofenergy reserves of cells since it can penetrate to miofibrillae,stimulate the symthesis of nucleotides and raise the activity of anumber of Krebs cycle enzymes.

The claimed solution contains all the three components which, used inlarge quantities, can serves as independent cardioplegic agents (ionsK⁺, ions Mg²⁺ and a local anesthetic). However, the quantity of each ofsaid components in the claimed solution is considerably smaller thanrequired for cardioplegy should each of them be used independently (e.g.30-40 mM/l for potassium and magnesium chlorides; 10 mM/l or more forthe local anesthetic, whichever of them is used). Such concentrations ofthe above-mentioned ions and lidocaine as suggested in the disclosedsolution cause a reverse stabilization of excitable membranes ofcardiomiocytes and other cells and do not bring about their irreversibledamage after prolonged total ischemia and subsequent reperfusion.Smaller concentrations deny the possibility of reaching the expectedeffect, for example during conservation of living organs andcardioplegia while larger concentrations may lead to an irreversibledamage to cell membranes.

The utilization of the claimed solution prolongs successful conservationof living organs, for example up to 48 h.

The claimed solution is a colorless transparent liquid with a pH of7.3-7.8. It is nontoxic and easily dissolves in water.

This solution is produced by simple mixing of its components.

The disclosed solution was tested in experiments involving conservationof the heart of a donor dog followed by transplanting it to a recipientdog. All in all there were 45 experiments whose results are summarizedin the Table.

The claimed solution was also tested in experiments involvingconservation of kidneys followed by transplanting them to mongrel whiterats.

The better understanding of the present invention it will be elucidatedby the following examples of its realization.

Example 1

The claimed solution contained components in the following ratio, mM/l:

    ______________________________________                                        Sodium chloride      110.0                                                    Potassium gluconate  16.0                                                     Magnesium sulphate   16.0                                                     Calcium gluconate    1.2                                                      Trihydroxymethylaminomethane                                                                       2.0                                                      Lidocaine            1.0                                                      Ethacysine           0.01                                                     Inosine              1.0                                                      Polyglucine          the balance                                              ______________________________________                                    

The claimed solution was prepared directly before the operation bysimple mixing of the components. Its osmolarity was 595 mOSm/l, and pHwas maintained at the level of 7.8. The solution was ozonized for 30 minto 400 mm Hg.

The donor was a 15-kg female mongrel dog. After intravenous injection ofsodium pentabarbital in a dose of 25 mg/kg bodyweight, the dog was putunder artificial respiration, at a breathing rate of 12 per minute,volume 200 ml. The rib cage was dissected sinistrally and dextrallybetween the third and fourth ribs by a median incision. The main vesselswere extracted, the superior cara vein and aorta were cannulated andligated. The heart-and-lung preparation was extracted from the organism.The trachea was reintubated and the heart-and-lung preparation wastransferred into a flack. The claimed cardioplegic solution was used forwashing and the liquid flowing off from the heart was collected in acontainer. The heart was stopped by the claimed cardioplagic solution inone minute's time. Then the heart was extracted, transferred into a300-ml container filled with the cardioplagic solution, and placed intoa refrigerator for 48 h at 7° C.

On expiration of 48 h the heart was removed from the refrigerator andsutured to the recipient dog in a heterotopic position. The recipientwas as 11-kg male mongrel dog. On connecting the veins and arteries, thecirculation of blood was slowly started in the transplanted heart whichwas immediately filled and began contracting. Contractions were soonfollowed by ventricular fibrillation which was arrested by adefibrillating discharge. Then the arterial pressure and ECG readingswere recorded, and a bioptic material was taken for anelectromicroscopic examination.

The autopsy of the dog's heart after 48-h conservation andtransplantation revealed the following changes. Against the backgroundof a good general conservation there were isnignificant changes of theelectrommicroscopic picture of the myocardial tissue in the form of amarble-like edema or a more global one with a moderate spread ofmitochondrion cristae or a certain lightening of the mitochondrialmatrix, a moderate degree of hyperchromatism or a normal conservation ofthe nuclear membrane. The changes also included an insignificant edemaof myocardial fibers with small myofibrilla divergence foci. Afterreperfusion of the donor's heart with the recipient's blood theabove-described minor changes discovered in the conserved heart vanishedcompletely within 30 minutes. Thus, the myocardial tissue was protectedby the solution against total ischemia damage within 48 h and subsequentreperfusion.

Example 2

    ______________________________________                                        Composition of the solution, mM/l:                                            sodium chloride       117.0                                                   Potassium gluconate   17.0                                                    Calcium gluconate     1.0                                                     Magnesium sulphate    32.0                                                    Trihydroxymethylaminomethane                                                                        2.5                                                     Lidocaine             1.66                                                    Ethacysine            0.02                                                    Inosine               2.0                                                     Polyglucine           the balance                                             ______________________________________                                    

The disclosed solution was prepared and administered as in Example 1 butwith pH=7.4.

The donor was a 5.4 kg female mongrel dog. General anesthesia:intravenous injection of sodium pentabarbital in a dose of 25 mg/kgbodyweight. The process of the operation was similar to that describedin Example 1.

The recipient was a 12-kg female mongrel dog. Transplantation of thedonor's heart to the recipient dog was as in Example 1 except that theheart was transplanted into a normotopic position. The heart resumedfunctioning after 10 minutes of slow perfusion with the recipient'sblood. Revification of the heart was accompanied by group atrial andventricular systoles and episodes of ventricular tachycardia. Therecorded parameters of ECG and arterial pressure did not reveal anydisturbance in heart functioning, dangerous to life. A bioptic samplewas taken for electromicroscopic examination. A necropsy of themyocardial tissue of the heart that was conserved for 25 h andtransplanted to the recipient revealed some changes similar to thoseobserved in Example 1 though by far less conspicuous, which vanishedcompletely after 30 minutes perfusion of the donor's heart with therecipient's blood. On the next day after recovering from anesthesia thedog felt normal; there were no changes in organs and systems except forsome atrial and ventricular extrasystoles recorded on ECG.

Example 3

    ______________________________________                                        Composition of the solution, mM/l:                                            Sodium chloride       100.0                                                   Potassium gluconate   12.0                                                    Calcium gluconate     2.0                                                     Magnesium sulphate    8.0                                                     Trihydroxymethylaminomethane                                                                        3.0                                                     Lidocaine             0.33                                                    Ethacysine            0.04                                                    Inosine               1.5                                                     Polyglucine           the balance                                             ______________________________________                                    

The solution was prepared as in Example 1 though with pH=7.3.

The donor was a 260-g nondescript white male rat. General etheranesthesia. Proximal laparotomy was made on the white line and theintestine was shifted dextrally to expose the surgery field. The kidneyartery and the entire kidney vein were carefully exposed. The ureter wasalso exposed and microsurgical forceps were applied to the kidney arteryand vein intersecting underneath. The donor's kidney was extracted fromthe operation wound and the kidney artery was cannulated. The kidney waswashed with the disclosed solution by introducing the latter into thekidney artery at the rate of 1 ml/min until a colorless fluid emergedfrom the kidney vein, then the kidney was transferred into a chamberfilled with the claimed solution. The chamber was placed into arefrigerator and stored for 26 h at 6° C.

The recipient was a 260-g white mongrel male rat. Its kidney was removedas described above and the intenstine was shifted aside to expose thesurgery field for transplantation of the donor's kidney. The kidneyvessels and the ureter were carefully extracted. Microforceps wereapplied to the kidney artery and vein and to the ureter intersectingunderneath. The second kidney was extracted and removed from the surgeryfield. The conserved kidney was carried from the refrigerator into theoperative field, localizing the kidney artery and vein and the ureter.Then anastomosis was applied between the donor's and recipient's kidneyarteries by individual interrupted sutures, using eight 10.0 ligatures.Then anastomosis of the kidney vein was formed by a continuous suture.The forceps were removed and blood circulation was restored in thetransplanted organ. The kidney filled evenly with a slight stenosis ofthe kidney vein wall. Anastomosis was straightened out by puncturing theinferior cava vein with the needle. Three sutures were applied to theureter. Execution of the anastomosis was slowed down by active dropwiseoutflow of urine. The wound was sutured up layerwise. On the next day,after recovery from anesthesia the condition of the rat with thetransplanted kidney conserved for 26 h was normal. So were thephysiological functions, and no disorders were observed in other organsand systems. A blood sample for analysis was taken from the tail vein.Dynamics of kidney weight were checked; the content ofadenosinetriphosphate diminished by 15% less than in the control testwith the kidney conserved by the cardioplegic solution used at the St.Thomas Hospital.

Industrial Applicability

The present invention can be utilized in medicine, particularly intransplantation of human patient's organs.

                                      TABLE                                       __________________________________________________________________________    Dynamics of Physiological Quotients in Animals                                after Transplantation of the Donor's Heart                                    (conservation time 24 h)                                                                       Postoperative period, h                                                       0                                                                             (immediately                                                     Physiological                                                                              after transp-                                                No. quotient     lantation)                                                                           4    24   48                                          __________________________________________________________________________    1.  Behavior quotients:                                                       1.1.                                                                              Orientation reflexes                                                                       none   changed                                                                            normal                                                                             normal                                      1.2.                                                                              Defense reflexes                                                                           none   changed                                                                            normal                                                                             normal                                      1.3.                                                                              Conditioned reflexes                                                                       none   changed                                                                            normal                                                                             normal                                      2.  Condition of organs                                                           and systems:                                                              2.1.                                                                              Motor activity                                                                             none   changed                                                                            changed                                                                            normal                                      2.2.                                                                              Uresis       changed                                                                              changed                                                                            normal                                                                             normal                                      2.3.                                                                              Digestion    changed                                                                              changed                                                                            changed                                                                            normal                                      3.  Body temperature                                                                           changed                                                                              changed                                                                            changed                                                                            normal                                      4.  Respiration rate                                                                           changed                                                                              changed                                                                            changed                                                                            normal                                      5.  Respiration depth                                                                          changed                                                                              changed                                                                            changed                                                                            normal                                      6.  Arterial pressure                                                                          changed                                                                              changed                                                                            normal                                                                             normal                                      7.  Heart rate   changed                                                                              changed                                                                            changed                                                                            normal                                      8.  Pulse        changed                                                                              changed                                                                            changed                                                                            normal                                      9.  Heart rhythm:                                                             9.1.                                                                              Atrial extraystoles                                                                        *      *    *    *                                           9.2.                                                                              Atrial flutter and                                                                         *      *    *    none                                            fibrillations                                                             9.3.                                                                              Ventricular extraystoles                                                                   *      *    *    *                                           9.4.                                                                              Sinus bradycardia                                                                          *      *    none none                                        9.5.                                                                              Sinus tachycardia                                                                          **     *    *    none                                        9.6.                                                                              Supraventricular                                                                           *      *    *    none                                            tachycardia                                                               9.7.                                                                              Ventricular tachycardia                                                                    *      none none none                                        9.8.                                                                              Ventricular fibrillation                                                                   *      none none none                                        9.9.                                                                              Blocks:                                                                   9.10.                                                                             Sinusal      *      *    none none                                        9.11.                                                                             Atrioventricular of various                                                                *      *    none none                                            degrees                                                                   9.12.                                                                             His bundle-branch block                                                                    *      none none none                                        10. ECG parameters:                                                           10.1.                                                                             Waves (duration and                                                           amplitude):                                                               10.2.                                                                             P            changed                                                                              normal                                                                             normal                                                                             normal                                      10.3.                                                                             Q            changed                                                                              normal                                                                             normal                                                                             normal                                      10.4.                                                                             R            changed                                                                              normal                                                                             normal                                                                             normal                                      10.5.                                                                             S            changed                                                                              normal                                                                             normal                                                                             normal                                      10.6.                                                                             T            changed                                                                              changed                                                                            changed                                                                            normal                                      10.7.                                                                             Intervals:                                                                10.8.                                                                             PQ           changed                                                                              normal                                                                             normal                                                                             normal                                      10.9.                                                                             QRS          changed                                                                              normal                                                                             normal                                                                             normal                                      10.10.                                                                            QT           changed                                                                              normal                                                                             normal                                                                             normal                                      10.11.                                                                            Fragment ST  changed                                                                              changed                                                                            changed                                                                            normal                                      11.1.                                                                             Physiological activity                                                        of analyzers:                                                             11.2.                                                                             Visual       changed                                                                              normal                                                                             normal                                                                             normal                                      11.3.                                                                             Acoustic     changed                                                                              normal                                                                             normal                                                                             normal                                      11.4.                                                                             Tactile      changed                                                                              normal                                                                             normal                                                                             normal                                      11.5.                                                                             Vestibular   changed                                                                              changed                                                                            normal                                                                             normal                                      11.6.                                                                             Gustatory    changed                                                                              changed                                                                            normal                                                                             normal                                      __________________________________________________________________________     Symbols:                                                                      *data unreliable (p 0.05)                                                     **data reliable (p 0.05)                                                 

We claim:
 1. A solution for conservation of living organs containingsodium chloride, salts of potassium, magnesium and calcium, a pHstabilizer and a solvent CHARACTERIZED in that it contains additionallya local anesthetic, phenothiazine and purine derivatives, potassium andcalcium gluconates as a substitute for potassium and calcium salts,magnesium sulphate as a substitute for the magnesium salt,trihydroxymethylaminomethane as a pH stabilizer, and polyglucine as asolvent in the following ratios of components, mM/l:

    ______________________________________                                        Sodium chloride      100.0-117.0                                              Potassium gluconate  12.0-17.0                                                Calcium gluconate    1.2-2.0                                                  Magnesium sulphate    8.0-32.0                                                Trihydroxymethylaminomethane                                                                       2.0-3.0                                                  Local anesthetic     0.33-2.66                                                Phenothiazine derivative                                                                           0.01-0.04                                                Purine derivative    1.0-2.0                                                  Polyglucine          the balance                                              ______________________________________                                    